The present invention relates to aryl imidate activated polyalkylene oxides having improved hydrolytic stability, and to water-soluble polyalkylene oxide conjugates prepared therefrom.
The conjugation of water-soluble polyalkylene oxides with useful molecules such as proteins and polypeptides is well known. The coupling of peptides and polypeptides to polyethylene glycol (PEG) and similar water-soluble polyalkylene oxides is disclosed by U.S. Pat. No. 4,179,337 to Davis et al.
Davis et al. discloses that physiologically active polypeptides modified with PEG exhibit dramatically reduced immunogenicity and antigenicity. Also, the polyalkylene oxide conjugates, when injected into a living organism, have been shown to remain in the bloodstream considerably longer than the corresponding native proteins. Accordingly, a number of polyalkylene oxide conjugated therapeutic proteins have been developed exhibiting reduced immunogenicity and antogenicity and longer clearance times, while retaining a substantial portion of the protein's physiological activity. Significant polyalkylene oxide conjugated therapeutic proteins include tissue plasminogen activator, insulin, interleukin II and hemoglobin.
The utility of polyalkylene oxide conjugation is not limited to the modification of proteins and polypeptides. Activated polyalkylene oxides will react with essentially any nucleophile. The coupling of polyalkylene oxides with oligonucleotides is disclosed by U.S. Pat. No. 4,904,582 to Tullis. U.S. Pat. No. 5,160,734 discloses sustained release formulations of polyalkylene oxides coupled with dihydropyridine calcium channel blockers.
To conjugate polyalkylene oxides, the hydroxyl end-groups of the polymer must first be converted into, that is, substituted with, reactive functional groups. This process is frequently referred to as "activation" and the product is called an "activated polyalkylene oxide."
Until recently, covalent attachment of the polyalkylene oxide to an appropriate nucleophile was effected by activated polyalkylene oxides such as polyalkylene oxide succinoyl-N-hydroxy succinate, as disclosed by Abuchowski et al., Cancer Biochem. Biophys., 7, 175-86 (1984). This polyalkylene oxide derivative is desirable because it is reactive under mild conditions.
A shortcoming associated with this derivative, however, is the fact that it is relatively hydrolytically unstable when no nucleophile is present. Recently, in U.S. Pat. No. 5,122,614, polyalkylene oxide-N-succinimide carbonates were disclosed having improved hydrolytic stability over the polyalkylene oxide succinoyl succinates. Even so, these active esters undergo hydrolysis under the pH conditions necessary to deprotonate the epsilon-NH.sub.2 groups of polypeptide lysines for conjugation, which subject the activated polyalkylene oxide to hydroxyl attack. This does not affect the reaction end product, other than to reduce its yield. While reduced yields ordinarily affect product cost, the hydrolysis becomes even more costly for several reasons. Firstly, reaction mixtures cannot be prepared significantly in advance. Additional purification of the end product is required to remove the hydrolytic degradation products. Furthermore, the reduction in yield is compensated for by increasing the amount of activated polyalkylene oxide starting material. This increases the viscosity of the reaction mixture, thereby further increasing the processing cost, and potentially interferes with downstream purification of the polymer and conjugate.
There remains a need for hydrolytically stable activated polyalkylene oxides. One group of newly developed polyalkylene oxides is the polyalkylene oxide alkyl imidates of U.S. Pat. No. 4,791,192. However, Hunter et al., J. Amer. Chem. Soc., 84, 3491-504 (1962) and Browne et al., Biochem. and Biophys. Res. Comm., 67(1), 126-32 (1975) studied the use of simple alkyl imidates to modify the primary amines of proteins and other small molecules and reported the alkyl imidates to be hydrolytically unstable at protein reaction conditions. Hunter et al., however, reported simple aryl imidates to be hydrolytically stable. Aryl imidate activated polyalkylene oxides are unreported.